Summary of Work: Exogenous DNA damaging agents such as cisplatinin, nitrogen mustard, and psoralen create interstrand DNA crosslinks. Such non-coding lesions must be repaired to ensure accurate replication of the genome and viability of the organism. The Drosophila mus308 mutation, which confers hypersensitivity to nitrogen mustard but not the monofunctional agent methyl-methane sulfonate, identified a DNA polymerase likely involved in processing DNA crosslinks. Based on homology to the Drosophila mus308 gene and another Family A DNA polymerase, DNA polymerase g, we cloned and expressed the cDNA for human DNA polymerase q. Amino acid sequence alignments predict DNA polymerase, ATP binding and/or hydrolysis, and 3' to 5' exonuclease functions for this enzyme. To verify these activities and to begin to characterize the role of this polymerase in DNA repair, we have generated a series of point mutations and deletions within the relevant motifs. Mutant and truncated proteins were purified from baculoviral infected insect cells. The substrate specificity and accuracy of DNA synthesis in vitro were determined. Unlike many of the other newly discovered DNA polymerases, polymerase theta synthesizes DNA with high fidelity.